Testosterone induces phosphorylation of CREB and ERK in primary rat Sertoli cells. (A) Sertoli cells were stimulated with ethanol (vehicle), estradiol (E2), and testosterone (T) at the concentrations shown or ovine FSH (100 ng/ml) for 15 min. Western immunoblotting of whole cell extracts was performed sequentially with antisera specifically recognizing CREB phosphorylated on serine 133 (P-CREB), P-ERK1/2, and all forms of ERK. Fractionation and probing of the samples was repeated by using an antiserum recognizing all forms of CREB. P-CREB and P-ERK1/2 levels were normalized to overall CREB and ERK1/2 expression, and the normalized P-CREB and P-ERK1/2 levels for vehicle-treated cells were arbitrarily set equal to 1. The mean fold induction (±SE) of hormone-stimulated P-CREB (▪) and P-ERK1/2 (□) over vehicle-treated cells for five (P-CREB) and four (P-ERK1/2) independent experiments is reported in the graph to the right. Values that are significantly different from vehicle-treated controls (P < 0.05) are indicted with an asterisk (*). (B) Sertoli cells were stimulated with ethanol for 15 min (0) or 100 nM R1881 for the times shown. Western analyses were performed as in A. The mean (±SE) relative levels of P-CREB (▪) and P-ERK1/2 (□) normalized for CREB and ERK1/2 expression are provided for four (P-CREB) and six (P-ERK1/2) independent experiments with statistical analysis as described in A. No changes in protein expression were observed over 12 h with vehicle (EtOH) controls (data not shown). (C) Sertoli cells were treated for 60 min with EtOH (vehicle), estradiol (E2), RU 5020, or testosterone (T). Western analyses were performed for P-CREB and CREB. The data shown are representative of three independent experiments. (D) Sertoli cells were pretreated with DMSO, actinomycin D (Actino D, 50 μg/ml), or puromycin (10 μg/ml) for 2 h before stimulation with EtOH vehicle (–) or 100 nM testosterone (+). Western blot analyses of P-CREB and CREB levels are shown. The blot shown is representative of at least three experiments.

Testosterone-induced phosphorylation of CREB is mediated by means of MAP kinase. (A) Sertoli cells were treated with FSH (100 ng/ml), testosterone (100 nM), or ethanol (vehicle) for 15 min. cAMP secreted into the media was measured by RIA. The data shown represent the mean levels of cAMP (±SE) for five observations. (B) DMSO (vehicle), PD98059 (PD, 50 μM), or wortmannin (Wort, 100 nM) were added to Sertoli cell cultures 1 h before stimulation with EtOH (–) or testosterone (+) (100 nM) for 15 min. Western immunoblotting was performed as in Fig. 1. Fractionization and probing of samples was repeated by using an antiserum recognizing phosphorylated protein kinase B, a target for wortmannin-sensitive phosphoinositide 3-kinase. The mean fold induction (±SE) of P-CREB levels over that of vehicle-treated cells is shown for three experiments.

RNA interference knock-down of AR expression inhibits androgen-mediated CREB phosphorylation. (A) Sertoli cells were pretreated with ICI 182,780 (ICI) or flutamide (Flut) (1 μM) for 4 h as indicated before stimulation with EtOH (Veh) or 100 nM testosterone (T) for 60 min. The western analysis shown of phosphorylated CREB and all forms of CREB is representative of three independent experiments. (B) Sertoli cells transfected with control luciferase (Luc) or AR siRNA oligonucleotides (20 nM) were stimulated 3 days after transfection with EtOH (vehicle) or 100 nM testosterone (T) for 15 min. Whole cell lysates were subjected to Western analysis as in Fig. 1. For each condition, the mean fold induction and SD for P-CREB expression over that of vehicle-treated cells transfected with luciferase siRNA is reported (n = 2). (C) Sertoli cells were transfected with luciferase (Left) or AR siRNA oligonucleotides (Right) (20 nM). Three days after transfection, immunofluorescence analysis was performed with AR antiserum and Alexa 488-conjugated secondary antiserum.

Testosterone induces CREB phosphorylation in Sertoli cells derived from wild-type but not AR-defective tfm mutant rats. (A) A portion of the sequencing results after PCR amplification of AR genomic DNA from wild-type and tfm rats is shown. The arrows denote the 1-bp difference in the sequences for AR. (B) A DIC image of Sertoli cells from 15-day-old tfm rats stained for peritubular cell-specific alkaline phosphatase activity after 3 days in culture shows that peritubular cells account for <5% of the culture. (C) The relative [³H]R1881-binding activities of wild-type (wt) and tfm Sertoli cells were determined by hormone-binding assay. The data shown represent the means (±SE) of three independent experiments. (D) Sertoli cell cultures from wild-type or tfm rats were stimulated with ethanol vehicle (V) or 100 nM testosterone (T) for 15 min. Whole cell extracts were subjected to Western analysis with antisera specific for P-CREB followed by reprobing with antisera against all CREB isoforms. (E) The relative mean fold inductions (±SE) of P-CREB levels for testosterone-stimulated cells versus ethanol-treated cells are shown for the studies performed in D (n = 4). (F) Sertoli cells derived from tfm rats were infected in duplicate with adenoviral vectors expressing β-galactosidase or AR and then stimulated 3 days later with EtOH (V) or 100 nM testosterone (T). CREB and P-CREB levels were determined by Western blot.

Androgen induces expression of endogenous CREB mRNA. RNA isolated from Sertoli cells stimulated with EtOH vehicle (–) or 100 nM R1881 (+) for 24 h (A) or 6 h (B) as indicated was used in reverse transcriptase reactions employing gene-specific primers. Reverse transcriptase reactions were performed in the absence (–) and presence (+) of reverse transcriptase enzyme (RT). PCR analysis was performed by using primers designed to amplify portions of CREB, LDH-A, Egr1, or GAPDH cDNAs. The cDNAs from the reverse transcriptase reactions were amplified for 25 cycles (CREB, LDH, and EGR1) or 20 cycles (GAPDH), and the resulting products were fractionated on agarose gels. The data shown are representative of more than three experiments. The migration of DNA size markers is shown to the left.